DESCRIPTION (Adapted from applicant's description): The aim of this pilot project is to identify the molecular defect in two patients with a severe combined immunodeficiency (SCID) syndrome. The patients are brothers born to consanguineous parents, thus the defect is likely to be a recessive mutation in a single gene. The patients' T cells exhibit a pronounced defect in capacitative (store-operated) Ca2+ entry; in consequence, they are unable to activate the calcineurin-regulated transcription factor NFAT (nuclear factor of activated T cells) and show a marked deficiency in transcription of cytokine genes. The underlying molecular defect likely involves a subunit of the capacitative Ca2+ entry channel (CRAC), or a component of the signaling pathway that leads to CRAC channel opening. The mechanisms of store-operated Ca2+ entry are essentially unknown but are likely to be conserved in many if not all non-excitable cell types, and our studies will therefore be of general interest to cell biologists as well as immunologists. The investigators will take two major approaches to identify the gene product that is defective in the patients' T cells: (i) Trapping the mutant gene by retroviral insertion in parental T cells. T cell lines from the heterozygous parents will be subjected to genome-wide retroviral insertion (gene-trapping) using a retroviral vector designed so that it can be cleanly excised from its site of integration in the genome. Mutant T cells defective in store-operated Ca2+ entry will be isolated from the population of infected cells. Cells in which retroviral excision reverts the mutant phenotype will be analyzed to determine the site of integration. Gene(s) identified by this process are strong candidates for the wildtype allele for which the parental cells are heterozygous. (ii) Complementation cloning. A microcell-mediated chromosome transfer (MMCT) approach will be used to identify human chromosomal regions that complement the capacitative calcium entry defect in the patients' T cells. Deletion mapping using spectral karyotyping and microsatellite markers will be used to further narrow down the chromosomal region involved in the defect and candidate genes in this region will be evaluated.